SummaryWe describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

The cereal caryopsis is a complex tissue in which maternal and endosperm tissues follow distinct but coordinated developmental programs. Because of the hexaploid genome in wheat (Triticum aestivum), the identification of genes involved in key developmental processes by genetic approaches has been difficult. To bypass this limitation, we surveyed 888 genes that are expressed during caryopsis development using a novel high-throughput mRNA in situ hybridization method. This survey revealed novel distinct spatial expression patterns that either reflected the ontogeny of the developing caryopsis or indicated specialized cellular functions. We have identified both known and novel genes whose expression is cell cycle-dependent. We have identified the crease region as important in setting up the developmental patterning, because the transition from proliferation to differentiation spreads from this region to the rest of the endosperm. A comparison of this set of genes with the rice (Oryza sativa) genome shows that approximately two-thirds have rice counterparts but also suggests considerable divergence with regard to proteins involved in grain filling. We found that the wheat genes had significant homology with 350 Arabidopsis thaliana genes. At least 25 of these are already known to be essential for seed development in Arabidopsis, but many others remain to be characterized.

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Small nucleolar RNAs (snoRNAs) are involved in many aspects of rRNA processing and maturation. In animals and yeast, a large number of snoRNAs are encoded within introns of protein-coding genes. These introns contain only single snoRNA genes and their processing involves exonucleolytic release of the snoRNA from debranched intron lariats. In contrast, some U14 genes in plants are found in small clusters and are expressed polycistronically. An examination of U14 flanking sequences in maize has identified four additional snoRNA genes which are closely linked to the U14 genes. The presence of seven and five snoRNA genes respectively on 2.05 and 0.97 kb maize genomic fragments further emphasizes the novel organization of plant snoRNA genes as clusters of multiple different genes encoding both box C/D and box H/ACA snoRNAs. The plant snoRNA gene clusters are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. The lack of exon sequences between the genes suggests that processing of polycistronic pre-snoRNAs involves endonucleolytic activity. Consistent with this, U14 snoRNAs can be processed from both non-intronic and intronic transcripts in tobacco protoplasts such that processing is splicing independent.